CN110954585B - Differential sensing of biological field effect transistor sensors - Google Patents

Abstract

Embodiments of the present invention relate to differential sensing of a bio-field effect transistor sensor, a method of manufacturing a bio-field effect transistor sensor, and a sensing method thereof. The present disclosure provides a sensor array including a semiconductor substrate, a first plurality of FET sensors, and a second plurality of FET sensors. Each of the FET sensors includes: a channel region between a source region and a drain region in the semiconductor substrate and under a gate structure disposed on a first side of the channel region; and a dielectric layer disposed on a second side of the channel region opposite the first side of the channel region. A first plurality of capture reagents is coupled to the dielectric layer over the channel regions of the first plurality of FET sensors and a second plurality of capture reagents is coupled to the dielectric layer over the channel regions of the second plurality of FET sensors.

Description

Differential sensing of biological field effect transistor sensors

Technical Field

Embodiments of the present invention relate to differential sensing of biological field effect transistor sensors.

Background

Biosensors are devices for sensing and detecting biomolecules and operate based on electronic, electrochemical, optical and mechanical detection principles. Biosensors comprising a transistor are sensors of electrical charge, photon and mechanical properties of an electrical biological entity or biological molecule. Detection may be performed by detecting the biological entity or the biological molecule itself or through interactions and reactions between the specified reactants and the biological entity/biological molecule. These biosensors can be manufactured using semiconductor processes, can rapidly convert electrical signals, and can be easily applied to Integrated Circuits (ICs) and microelectromechanical systems (MEMS).

Disclosure of Invention

An embodiment of the invention relates to a sensor array comprising: a semiconductor substrate; a first plurality of FET sensors, each of the first plurality of FET sensors comprising: a first channel region between a source region and a drain region in the semiconductor substrate and under a gate structure disposed on a first side of the first channel region, a dielectric layer disposed on a second side of the first channel region opposite the first side of the first channel region, and a first plurality of capture reagents coupled to the dielectric layer over the first channel region; and a second plurality of FET sensors, each of the second plurality of FET sensors comprising: a second channel region located between a source region and a drain region in the semiconductor substrate and below a gate structure disposed on a first side of the second channel region, the dielectric layer disposed on a second side of the second channel region opposite the first side of the second channel region, and a second plurality of capture reagents coupled to the dielectric layer over the second channel region, wherein the second plurality of capture reagents is different from the first plurality of capture reagents, wherein the first plurality of FET sensors and the second plurality of FET sensors are arranged in a two-dimensional array and coupled to a common reference electrode.

An embodiment of the invention relates to a method comprising: depositing a first solution droplet containing a first plurality of capture reagents over a first plurality of FET sensors formed in a semiconductor substrate such that the first plurality of capture reagents bind to a dielectric layer on a first surface of the semiconductor substrate in a first plurality of openings arranged over the first plurality of FET sensors; depositing a second solution droplet containing a second plurality of capture reagents over a second plurality of FET sensors formed in the semiconductor substrate such that the second plurality of capture reagents bind to the dielectric layer on the first surface of the semiconductor substrate in a second plurality of openings arranged over the second plurality of FET sensors, the second plurality of capture reagents being different from the first plurality of capture reagents; forming a fluid delivery system configured to introduce a target solution over the first plurality of FET sensors and the second plurality of FET sensors; coupling a plurality of first gate structures of the first plurality of FET sensors to a controller configured to apply a first voltage to the plurality of first gate structures, wherein the plurality of first gate structures are located on a second surface of the semiconductor substrate opposite the first surface of the semiconductor substrate; coupling a plurality of second gate structures of the second plurality of FET sensors to the controller, the controller configured to apply a second voltage to the plurality of second gate structures, wherein the plurality of second gate structures are located on a second surface of the semiconductor substrate opposite the first surface of the semiconductor substrate; and coupling the first plurality of FET sensors and the second plurality of FET sensors to a readout circuit configured to determine the presence of one or more target analytes in the target solution based on the application of at least one of the first voltage and the second voltage.

An embodiment of the invention relates to a method of manufacturing a biosensor system, the method comprising: depositing a solution containing a plurality of capture reagents over a plurality of FET sensors formed in a semiconductor substrate such that the plurality of capture reagents bind to a dielectric layer on a first surface of the semiconductor substrate in a plurality of openings disposed over the plurality of FET sensors; forming a fluid delivery system configured to introduce a second solution over the plurality of FET sensors such that one or more cells in the second solution bind to the capture reagent bound to the dielectric layer of the plurality of FET sensors; coupling a plurality of gate structures of the plurality of FET sensors to a controller, the controller configured to apply a first voltage and a second voltage to the plurality of gate structures, wherein the plurality of gate structures are located on a second surface of the semiconductor substrate opposite the first surface of the semiconductor substrate, and wherein the second voltage is applied a given period of time after the application of the first voltage; and coupling the plurality of FET sensors to a readout circuit configured to measure first and second current responses of the plurality of FET sensors based on the application of the first and second voltages, wherein the readout circuit is further configured to determine a growth characteristic of the one or more cells based on a comparison between the first and second current responses.

Drawings

Aspects of the disclosure are best understood from the following detailed description when read in conjunction with the accompanying drawing figures. It should be noted that the various devices are not necessarily drawn to scale according to standard industry practice. In fact, the dimensions of the various devices may be arbitrarily increased or reduced for clarity of discussion.

FIG. 1 illustrates components of a sensing device according to some embodiments.

FIG. 2 depicts a cross-sectional view of an example dual gate backside sense FET sensor in accordance with some embodiments.

Fig. 3 is a circuit diagram of a plurality of FET sensors configured in an example addressable array, according to some embodiments.

Fig. 4 is a circuit diagram of an example addressable array of dual gate FET sensors and heaters, according to some embodiments.

FIG. 5 depicts a cross-sectional view of an example dual gate backside sense FET sensor in accordance with some embodiments.

Fig. 6A and 6B illustrate the use of a dual gate backside sense FET sensor as a pH sensor in accordance with some embodiments.

Fig. 7 depicts a cross-sectional view of an exemplary dual gate backside sensing bioFET detecting the presence of cells or other microorganisms, according to some embodiments.

FIG. 8 depicts an example metabolic pathway of glucose.

FIG. 9 illustrates a sense array of FET sensors according to some embodiments.

FIG. 10 depicts capturing analytes using a sensing array according to some embodiments.

FIG. 11 illustrates capturing different analytes using a sensing array according to some embodiments.

FIG. 12 illustrates monitoring cell growth using a sensing array according to some embodiments.

FIG. 13 illustrates placement of different capture reagents across a sensing array according to some embodiments.

FIG. 14 is a flow chart of an example method of performing sensing with a sensor array.

FIG. 15 is a flow chart of another example method of performing sensing with a sensor array.

Detailed Description

The following disclosure provides different embodiments, or examples, for implementing different features of the disclosure. Specific examples of components and arrangements are described below to simplify the present disclosure. Of course, these are merely examples and are not intended to be limiting. For example, forming a first device over a second device in the description below may include embodiments in which the first device and the second device are formed in direct contact, and may also include embodiments in which additional devices may be formed and/or placed between the first device and the second device such that the first device and the second device may not be in direct contact. Additionally, the present disclosure may repeat element symbols and/or letters in the various examples. This does not itself dictate a relationship between the various embodiments and/or configurations discussed.

Moreover, for ease of description, spatially relative terms such as "below …," "below …," "lower," "above …," "upper," and the like may be used herein to describe one element or device's relationship to another (other) element or device as depicted in the figures. Spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. The apparatus may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments according to the present disclosure; methods, apparatus, and materials are now described. For the purposes of describing and disclosing the materials and methods reported in the disclosure that may be used in connection with the present disclosure, all patents and publications mentioned herein are incorporated herein by reference.

As used herein, the acronym "FET" refers to a field effect transistor. One type of FET is known as a "metal oxide semiconductor field effect transistor" (MOSFET). A MOSFET may be a planar structure built in and on a planar surface of a substrate, such as a semiconductor wafer. MOSFETs may also have three-dimensional, fin-based structures.

The term "bioFET" refers to a FET that comprises a layer of capture reagent that acts as a surface receptor to detect the presence of a target analyte of biological origin. According to some embodiments, the bioFET is a field effect sensor with a semiconductor sensor. One advantage of a bioFET is the prospect of label-free operation. In particular, biofets can avoid expensive and time-consuming labelling operations, such as labelling analytes with, for example, fluorescent or radioactive probes. One particular type of bio fet described herein is a "dual gate backside sensing bio fet". The analyte for bioFET detection may be of biological origin, such as, for example, but not limited to, a protein, a carbohydrate, a lipid, a tissue fragment, or a portion thereof. biofets may be part of a more general class of FET sensors that can also detect chemical compounds; this type of bioFET is known as a "ChemFET" or any other element. biofets can also detect ions, such as protons or metal ions; this type of bioFET is known as an "ISFET". The present disclosure is applicable to all types of FET-based sensors ("FET sensors"). One particular type of FET sensor described herein is a "dual gate backside sense FET sensor" (DG BSS FET sensor).

"S/D" refers to the source/drain junctions that form two of the four terminals of the FET.

The expression "high k" refers to a high dielectric constant. In the semiconductor device structure and manufacturing process arts, high k refers to a material greater than SiO ₂ A dielectric constant of (i.e., greater than 3.9).

As used herein, the term "perpendicular" means nominally perpendicular to the surface of the substrate.

The term "analysis" generally refers to a procedure or step involving physical, chemical, biochemical, or biological analysis, including but not limited to characterization, testing, measurement, optimization, separation, synthesis, addition, filtration, solubilization, or mixing.

The term "assay" generally refers to procedures or steps involving analysis of chemical or target analytes and includes, but is not limited to, cell-based assays, biochemical assays, high-throughput measurement and screening, diagnostic assays, pH determinations, nucleic acid hybridization assays, polymerase activity assays, nucleic acid and protein sequencing, immunoassays (e.g., antibody-antigen binding assays, ELISA and iqPCR), bisulfite methylation assays for detecting methylation patterns of genes, protein assays, protein binding assays (e.g., protein-protein, protein-nucleic acid and protein-ligand binding assays), enzyme assays, coupled enzyme assays, kinetic measurements (e.g., protein folding kinetics and enzyme reaction kinetics), enzyme inhibitor and activator screening, chemical fluorescence and electrochemical fluorescence assays, fluorescent polarization and anisotropy assays, absorbance and colorimetric assays (e.g., bradford assays, lowry assays, hartree-Lowry assays, biuret assays and Biuret assays), chemical assays (e.g., for detecting environmental pollutants (poltan and polymers), nanoparticles or polymers), and drug discovery, genome-based assays, gene profiling, genetic profiling, non-invasive clinical analysis, non-invasive analysis, non-clinical analysis, and non-invasive analysis of cancer, clinical analysis, and non-invasive analysis. The apparatus, systems, and methods described herein may use or employ one or more of these assays for use with any FET sensor description design.

The term "liquid biopsy" generally refers to a biopsy sample obtained from a bodily fluid of a subject as compared to a tissue sample of the subject. The ability to perform an assay using a body fluid sample is generally more desirable than using a tissue sample. Less invasive methods using body fluid samples have a broad impact on patient welfare, ability to monitor longitudinal disease, and ability to obtain expression profiles even when tissue cells are not readily accessible, such as in the prostate. Assays for detecting target analytes in liquid biopsy samples include, but are not limited to, those described above. As a non-limiting example, a liquid biopsy sample may be subjected to a Circulating Tumor Cell (CTC) assay.

For example, capture reagents (e.g., antibodies) immobilized on FET sensors can be used to detect target analytes (e.g., tumor cell markers) in liquid biopsy samples using CTC assays. CTCs are cells that flow from a tumor into the vasculature and circulate in, for example, the blood stream. Typically, CTCs are present in the circulation at low concentrations. To determine CTCs, CTCs are enriched from the patient's blood or plasma by various techniques known in the art. CTCs may be stained for specific markers using methods known in the art, including but not limited to methods based on cytometry (e.g., flow cytometry) and IHC-based methods. For the devices, systems, and methods described herein, CTCs may be captured or detected using a capture reagent, or nucleic acids, proteins, or other cellular environments from CTCs may be targeted to target analytes for binding to or detection by the capture reagent.

For example, when detecting a target analyte on or from CTCs, an increase in expression or inclusion of a target analyte of CTCs may help identify the subject as having a cancer that is likely to be responsive to a particular therapy (e.g., a therapy associated with the target analyte) or allow optimization of a therapeutic regimen, e.g., having antibodies to the target analyte. CTC measurement and quantification may provide information about, for example, tumor stage, response to therapy, disease progression, or a combination thereof. Information obtained from detecting target analytes on CTCs can be used, for example, as prognostic, predictive, or pharmacodynamic biomarkers. In addition, CTC assays of liquid biopsy samples may be used alone or in combination with additional tumor marker assays of solid biopsy samples.

The term "identify" generally refers to a procedure that determines the identity of a target analyte based on its binding to a capture reagent of known identity.

The term "measurement" generally refers to a procedure that determines the amount, quantity, mass, or property of a target analyte based on its binding to a capture reagent.

The term "quantification" generally refers to a procedure for determining the amount or concentration of a target analyte based on its binding to a capture reagent.

The term "detection" generally refers to a procedure for determining the presence or absence of a target analyte based on its binding to a capture reagent. Detection includes, but is not limited to, identification, measurement, and quantification.

The term "chemical" refers to a substance, compound, mixture, solution, emulsion, dispersion, molecule, ion, dimer, macromolecule, such as a polymer or protein, biomolecule, precipitate, crystal, chemical functional group or group, particle, nanoparticle, reagent, reaction product, solvent, or fluid, any of which may exist in a solid, liquid, or gaseous state and may be the subject of analysis.

The term "reaction" refers to a physical, chemical, biochemical or biological transformation involving at least one chemical species and generally involving (in the case of chemical, biochemical and biological transformations) the cleavage or formation of one or more bonds, such as covalent, non-covalent, van der Waals, hydrogen or ionic bonds. The term includes chemical reactions such as, for example, synthesis reactions, neutralization reactions, decomposition reactions, metathesis reactions, reduction-oxidation reactions, precipitation, crystallization, combustion reactions, and polymerization reactions, as well as covalent and non-covalent bonding, phase changes, color changes, phase formation, crystallization, dissolution, light emission, light absorption or emission property changes, temperature changes or heat absorption or emission, conformational changes, and folding or unfolding of macromolecules (e.g., proteins).

As used herein, a "capture reagent" is a molecule or compound capable of binding a target analyte, which may be directly or indirectly attached to a substantially solid material. The capture reagent may be a chemical, and in particular any substance for which a naturally occurring target analyte (e.g., antibody, polypeptide, DNA, RNA, cell, virus, etc.) is present or for which a target analyte may be prepared, and the capture reagent may bind to one or more target analytes in an assay. The capture reagent may be non-naturally occurring or naturally occurring, and if naturally occurring, may be synthesized in vivo or in vitro.

As used herein, a "target analyte" is a substance to be detected in a test sample using embodiments of the present disclosure. The target analyte may be a chemical, and in particular any substance for which a naturally occurring capture reagent (e.g., antibody, polypeptide, DNA, RNA, cell, virus, etc.) is present or for which a capture reagent may be prepared, and the target analyte may bind to one or more capture reagents in the assay. "target analyte" also includes any antigenic material, antibodies, and combinations thereof. Target analytes may include proteins, peptides, amino acids, carbohydrates, hormones, steroids, vitamins, drugs (including those administered for therapeutic purposes as well as those administered for illicit purposes), bacteria, viruses, and metabolites of antibodies or antibodies to any of the foregoing.

As used herein, "test sample" means a composition, solution, substance, gas, or liquid containing a target analyte to be detected and assayed using embodiments of the present disclosure. The test sample may contain other components in addition to the target analyte, may have physical properties of a liquid or gas, and may be of any size or volume, including, for example, a moving liquid or gas stream. The test sample may contain any substance other than the target analyte, as long as the other substance does not interfere with the binding of the target analyte to the capture reagent or the specific binding of the first binding member (binding member) to the second binding member. Examples of test samples include, but are not limited to, naturally occurring samples, non-naturally occurring samples, or combinations thereof. Naturally occurring test samples may be synthetic or may be synthetic. Naturally occurring test samples include bodies or body fluids isolated from any place within or on the body of the subject, including, but not limited to, blood, plasma, serum, urine, saliva or sputum, spinal fluid, cerebrospinal fluid, pleural effusion, nipple aspirate fluid, lymph fluid, respiratory fluid, intestinal fluid and genitourinary fluid, tears, saliva, breast milk, lymphatic system fluid, semen, organ system fluid, ascites, tumor cyst fluid, amniotic fluid, and combinations thereof, as well as environmental samples such as groundwater or wastewater, soil extracts, air and pesticide residues, or food related samples.

The substances detected may include, for example, nucleic acids (including DNA and RNA), hormones, different pathogens (including biological agents that cause disease or illness to their host, such as viruses (e.g., H7N9 or HIV), protozoa (e.g., malaria caused by plasmodium), or bacteria (e.g., escherichia coli or tuberculosis)), proteins, antibodies, various drugs or treatments, or other chemical or biological substances, including hydrogen or other ions, nonionic molecules or compounds, polysaccharides, small chemical compounds (e.g., members of chemical combinatorial libraries, and the like). The detected or determined parameters may include, but are not limited to, for example, pH changes, lactose changes, concentration changes, particles per unit time (where a fluid flows over the device for a period of time to detect particles, such as sparse particles), and other parameters.

As used herein, the term "immobilized" when used with respect to, for example, a capture reagent includes attaching the capture reagent to a surface substantially at the molecular level. For example, adsorption techniques including non-covalent interactions (e.g., electrostatic forces, van der Waals forces, and dehydration of hydrophobic interfaces) and covalent bonding techniques (where functional groups or linkers facilitate attachment of capture reagents to the surface of the matrix material) may be used to immobilize the surface. The immobilization of the capture reagent to the surface of the matrix material may be based on the nature of the surface of the matrix, the medium carrying the capture reagent, and the nature of the capture reagent. In some cases, the substrate surface may first be modified to have functional groups bound to the surface. The functional group can then be bound to a biomolecule or biological or chemical substance to immobilize the functional group on the biomolecule or biological or chemical substance.

The term "nucleic acid" generally refers to a set of nucleotides linked to each other via phosphodiester bonds, and refers to naturally occurring nucleic acids to which naturally occurring nucleotides found in nature are linked, such as DNA comprising deoxynucleotides having any one of adenine, guanine, cytosine, and thymine linked to each other and/or RNA comprising ribonucleotides having any one of adenine, guanine, cytosine, and uracil linked to each other. Naturally occurring nucleic acids include, for example, DNA, RNA, and micrornas (mirnas). In addition, non-naturally occurring nucleotides and non-naturally occurring nucleic acids are within the scope of the nucleic acids of the present disclosure. Examples include cDNA, peptide Nucleic Acid (PNA), peptide nucleic acid with phosphate groups (PHONA), bridged nucleic acid/locked nucleic acid (BNA/LNA), and morpholino nucleic acid. Further examples include chemically modified nucleic acids and nucleic acid analogs such as methylphosphonate DNA/RNA, phosphorothioate DNA/RNA, phosphoramidate DNA/RNA, and 2' -O-methyl DNA/RNA. The nucleic acid comprises a modifiable nucleic acid. For example, the phosphate groups, sugar and/or base in the nucleic acid may be labeled as desired. Any substance known in the art for nucleic acid labeling can be used for labeling. Examples include, but are not limited to, radioisotopes (e.g., 32P, 3H, and 14C), DIG, biotin, fluorescent dyes (e.g., FITC, texas, cy, cy5, cy7, FAM, HEX, VIC, JOE, rox, TET, bodipy493, NBD, and TAMRA), and luminescent substances (e.g., acridinium esters).

As used herein, an aptamer refers to an oligonucleotide or peptide molecule that binds to a particular target molecule. The concept of using single-stranded nucleic acids (aptamers) as affinity molecules for protein binding was originally described in Ellington, andrew D and Jack W.Szostank, "in vitro selection of single-stranded DNA molecules folded into specific ligand binding structures" (Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures), "Nature" (1992): 850-852; "exponential enriched ligand system evolution" by Tuerk, craig and Larry Gold: RNA ligand to phage T4DNA polymerase (Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4DNA polymerase) "," Science (1990): 249.4968 (1990): 505-510 and is based on the ability of a short sequence to fold into a unique three-dimensional structure that binds a target with high affinity and specificity in the presence of a target. Ng, eugene WM et al, "Pegatanib, targeted anti-VEGF aptamer for ocular vascular disease" (a targeted anti-VEGF aptamer for ocular vascular disease), "Nature Reviews, drug Discovery", 5.2 (2006): 123 discloses that the aptamer is an oligonucleotide ligand selected for high affinity binding to a molecular target.

The term "protein" generally refers to a group of amino acids that are typically joined together in a specific sequence. The protein may be naturally occurring or non-naturally occurring. As used herein, the term "protein" includes amino acid sequences, as well as amino acid sequences that have been modified to contain functional groups or groups, such as sugars, polymers, metal organic groups, luminescent (or light-emitting) groups, functional groups or groups that enhance or participate in procedures such as intramolecular or intermolecular electron transfer, functional groups or groups that promote or induce a protein to assume a particular configuration or series of configurations, functional groups or groups that hinder or inhibit a protein to assume a particular configuration or series of configurations, functional groups or groups that induce, enhance or inhibit protein folding, or other functional groups or groups that are incorporated into an amino acid sequence and are intended to modify the chemical, biochemical or biological properties of the sequence. As used herein, proteins include, but are not limited to, enzymes, structural elements, antibodies, antigen-binding antibody fragments, hormones, receptors, transcription factors, electronic carriers, and other macromolecules involved in procedures such as cellular procedures or activities. Proteins can have up to four structural levels, including primary, secondary, tertiary, and quaternary structures.

As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that is capable of non-covalently, reversibly and in a specific manner binding to a corresponding antigen. For example, naturally occurring IgG antibodies are tetramers comprising at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain CL. VH and VL regions may be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDRs constitute about 15% to 20% of the variable domain. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). (Kuby, immunology, 4 th edition, chapter 4, w.h. freeman & co., new york, 2000).

The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-specific (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the present disclosure). Antibodies can be of any isotype/class (e.g., igG, igE, igM, igD, igA and IgY) or subclass (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2).

As used herein, the term "antigen binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization/destabilization, and spatial distribution). Examples of binding fragments include, but are not limited to, single chain Fv (scFv), camelbodies, disulfide bonded Fv (sdFv), fab fragments, F (ab') fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; a F (ab) 2 fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; fd fragment consisting of VH and CH1 domains; fv fragment consisting of the VL and VH domains of the single arm of the antibody; dAb fragments (Ward, e.sally et al, "binding Activity of antibody repertoire of individual immunoglobulin variable domains secreted from E.coli (Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli)", "Nature 341.6242 (1989): 544-546), which consist of VH domains; and isolating Complementary Determining Regions (CDRs), or other epitope-binding fragments of antibodies.

Furthermore, although the two domains of Fv fragments (VL and VH) are encoded by separate genes, they can be joined (using recombinant methods) by a synthetic linker that enables them to function as a Single protein chain, in which the VL and VH regions pair to form monovalent molecules (known as Single chain Fv ("scFv"); see, e.g., bird, robert E. Et al, "Single chain antigen-binding proteins"; science 242.4877 (1988): 423-427; and protein engineering of the "antibody binding sites" produced in E.coli; huston, james S. Et al; recovery of specific activity in the anti-digoxin Single chain Fv simulation (Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin Single-chain Fv analogue produced in Escherichia coli) "," Proc. Of U.S. Sci.5879-5883; these Single chain antibodies are also intended to be covered by the term "antigen binding fragments". These antigen binding fragments "are known to the user and are available in the same manner as the conventional art.

Antigen binding fragments may also be incorporated into single domain antibodies, large antibodies, mini-antibodies, single domain antibodies, intracellular antibodies, diabodies, trivalent antibodies, tetravalent antibodies, v-NARs, and bis-scFvs (see, e.g., holliger, philipp and Peter J.Hudson, "engineered antibody fragments and the rise of single domains (Engineered antibody fragments and the rise of single domains)", nature. Biotechnology (Nature Biotechnology) > 23.9 (2005): 1126). The antigen binding fragments may be grafted into the scaffold based on a polypeptide, such as fibronectin type III (Fn 3) (see U.S. patent No. 6,703,199, which describes fibronectin polypeptide monomers).

Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv fragments (VH-CH 1-VH-CH 1) that together with a complementary light chain polypeptide form a pair of antigen binding regions (Zapata, gerardo et al, "engineered linear F (ab ') 2fragments (Engineering linear F (ab') 2fragments for efficient production in Escherichia coli and enhanced antiproliferative activity) for efficient production and enhanced antiproliferative activity in E.coli)", "protein engineering, design and selection (Protein Engineering, design and Selection)" 8.10 (1995): 1057-1062 and U.S. Pat. No. 5,641,870).

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a polypeptide, including antibodies and antigen-binding fragments having substantially the same amino acid sequence or derived from the same gene source. This term also encompasses the preparation of antibody molecules of a single molecule composition. The monoclonal antibody compositions exhibit single binding specificity and affinity for a particular epitope.

The term "nanoparticle" refers to an atom, molecule or macromolecular particle of length scale, e.g., approximately 1nm to 100 nm. Novel and different properties and functions of nanoparticles are observed or developed at critical length scales of matter (e.g., such as below 100 nm). Nanoparticles can be used to build nano-scale structures and can be integrated into larger material components, systems, and architectures. In some embodiments, critical length scales involving novel properties and phenomena of nanoparticles may be less than 1nm (e.g., manipulating atoms of approximately 0.1 nm) or they may be greater than 100nm (e.g., nanoparticle reinforced polymers have unique features of approximately 200nm to 300nm depending on local bridge or bond variations between the nanoparticle and the polymer).

The term "nucleation composition" refers to a substance or mixture comprising one or more nuclei capable of growing crystals under conditions suitable for crystal formation. For example, the nucleating composition may be induced to undergo crystallization by evaporation, reagent concentration changes, addition of substances (e.g., precipitants), seeding with solid materials, mechanical agitation, or scraping of surfaces in contact with the nucleating composition.

The term "microparticle" refers to a cluster or agglomeration of particles, such as an atom, molecule, ion, dimer, polymer, or biomolecule. The particles may comprise a solid substance or be substantially solid, but they may also be porous or partially hollow. Which may contain a liquid or a gas. In addition, the microparticles may be homogeneous or heterogeneous; that is, it may comprise one or more substances or materials.

The term "polymer" means any substance or compound composed of two or more building blocks ("mers") repeatedly linked to each other. For example, a "dimer" is a compound in which two building blocks have been joined together. The polymer includes both polycondensates and addition polymers. Examples of polycondensates include polyamides, polyesters, proteins, wool, silk, polyurethanes, cellulose and silicones. Examples of addition polymers are polyethylene, polyisobutylene, polyacrylonitrile, poly (vinyl chloride) and polystyrene. Other examples include polymers with enhanced electrical or optical properties (e.g., nonlinear optical properties), such as conductive or photorefractive polymers. The polymer includes both linear and branched polymers.

Overview of exemplary biosensing devices

FIG. 1 depicts an overview of components that may be included in a biosensor system 100. The biosensor system 100 includes: a sensor array 102 having at least one sensing element for detecting a biological or chemical analyte; and a fluid delivery system 104 designed to deliver one or more fluid samples to the sensor array 102. The fluid delivery system 104 may be a microfluidic well positioned over the sensor array 102 to hold fluid over the sensor array 102. The fluid delivery system 104 may also include microfluidic channels for delivering various fluids to the sensor array 102. The fluid delivery system 104 may include any number of valves, pumps, chambers, channels designed to deliver fluid to the sensor array 102. The sensor array 102 may include a repeating layout of sensors across a surface. For example, the sensors may be arranged in a two-dimensional array of rows and columns across the surface.

According to some embodiments, readout circuitry 106 is provided to measure signals from the sensors in sensor array 102 and generate quantifiable sensor signals indicative of the amount of a particular analyte present in the target solution.

The controller 108 may be used to send and receive electrical signals to both the sensor array 102 and the readout circuitry 106 to perform biological or chemical sensing measurements. The controller 108 may also be used to send electrical signals to the fluid delivery system 104 to actuate, for example, one or more valves, pumps, or motors.

The sensor array 102 may include an array of bio fets, wherein one or more of the bio fets in the array are functionalized to detect a particular target analyte. Different ones of the sensors may be functionalized with different capture reagents to detect different target analytes. Further details regarding example designs of particular bio fets are provided below. The biofets may be arranged in a plurality of rows and columns, forming a two-dimensional sensor array. In some embodiments, each row of biofets is functionalized with a different capture reagent. In some embodiments, each column of biofets is functionalized with a different capture reagent. In some embodiments, different sectors of the 2-dimensional array are functionalized with different capture reagents.

The controller 108 may include one or more processing devices, such as a microprocessor, and may be programmable to control the operation of the readout circuitry 106 and/or the sensor array 102. The details of the controller 108 itself are not important to an understanding of the embodiments described herein. However, various electrical communications that may be transmitted and received from the sensor array 102 will be discussed in more detail below.

Dual gate backside FET sensor

Embodiments described herein relate to differentially measuring signals from one or more bio fet sensors or a bio fet sensor array to reduce common noise between the bio fet sensors. Achieving this involves controlling fluid delivery to two separate bio fet sensors or bio fet sensor arrays and differentially sensing measured signals from each of the bio fet sensors or bio fet sensor arrays. This particular paragraph describes an example bioFET sensor design that may be used in embodiments of the present application.

One example type of bioFET sensor that may be used in the sensor array 102 is a dual gate backside FET sensor. Dual gate backside FET sensors utilize semiconductor fabrication techniques and biological capture reagents to form an array sensor. While a MOSFET may have a single gate electrode connected to a single electrical node, a dual gate backside sense FET sensor has two gate electrodes, each of which is connected to a different electrical node. A first of the two gate electrodes is referred to herein as a "front side gate" and a second of the two gate electrodes is referred to herein as a "back side gate". Both the front side gate and the back side gate are configured such that in operation, each gate can be charged and/or discharged and thereby each affect the electric field between the source/drain terminals of the dual gate back side sense FET sensor. The front side gate is conductive, separated from the channel region by a front side gate dielectric, and configured to charge and discharge through the circuit to which it is coupled. The back-side gate is separated from the channel region by a back-side gate dielectric and includes a biofunctionalized sensing layer disposed on the back-side gate dielectric. The amount of charge on the backside gate depends on whether a biometric response has occurred. In operation of the dual gate backside sense FET sensor, the front side gate is charged to a voltage within a predetermined voltage range. The voltage on the front side gate determines the corresponding conductivity of the channel region of the FET sensor. A relatively small change in the charge on the backside gate changes the conductivity of the channel region. It is this conductivity change that is indicative of the biometric response.

One advantage of FET sensors is the prospect of label-free operation. In particular, FET sensors can avoid expensive and time-consuming labeling operations, such as labeling analytes with, for example, fluorescent or radioactive probes.

FIG. 2 depicts an example dual gate backside sense FET sensor 200 according to some embodiments. The dual gate backside sense FET sensor 200 includes a control gate 202, the control gate 202 being formed on a surface of a substrate 214 and separated from the substrate 214 by an intervening dielectric 215 placed on the substrate 214. An interconnect region 211 comprising a plurality of interconnect layers may be provided over one side of the substrate 214. The substrate 214 includes a source region 204, a drain region 206, and a channel region 208 between the source region 204 and the drain region 206. In some embodiments, the substrate 214 has a thickness between about 100nm and about 130 nm. The gate 202, source region 204, drain region 206, and channel region 208 may be formed using suitable CMOS process technology. The gate 202, source region 204, drain region 206, and channel region 208 form a FET. Isolation layer 210 is placed on the opposite side of substrate 214 from gate 202. In some embodiments, isolation layer 210 has a thickness of about 1 μm. In this disclosure, the side of the substrate 214 over which the gate 202 is placed is referred to as the "front side" of the substrate 214. Similarly, the side of the substrate 214 on which the isolation layer 210 is disposed is referred to as the "backside".

Openings 212 are provided in isolation layer 210. The opening 212 may be substantially aligned with the gate 202. In some embodiments, the opening 212 is larger than the gate 202 and may extend over multiple dual gate backside sense FET sensors. An interface layer (not shown) may be placed in the opening 212 on the surface of the channel region 208. The interface layer may be operable to provide an interface for locating and immobilizing one or more receptors for detecting biomolecules or biological entities. Further details regarding the interface layer are provided herein.

The dual gate backside sense FET sensor 200 includes electrical contacts 216 and 218 to the drain region 206 and the source region 204, respectively. A front side gate contact 220 may be made to the gate 202 and a back side gate contact 222 may be made to the channel region 208. It should be noted that the backside gate contact 222 need not physically contact the substrate 214 or any interface layer above the substrate 214. Thus, while FETs may use gate contacts to control conductance of a semiconductor (e.g., channel) between source and drain, dual gate backside sense FET sensor 200 allows a receiver to be formed on the side opposite gate 202 of the FET device to control conductance, while gate 202 provides another region to control conductance. Thus, the dual gate backside sense FET sensor 200 may be used to detect one or more specific biomolecules or biological entities in the surrounding environment and/or in the opening 212, as discussed in more detail using the examples herein.

The dual gate backside sense FET sensor 200 may be connected to: additional passive components such as resistors, capacitors, inductors, and/or fuses; other active components including p-channel field effect transistors (PFETs), n-channel field effect transistors (NFETs), metal Oxide Semiconductor Field Effect Transistors (MOSFETs), high voltage transistors, and/or high frequency transistors; other suitable components; or a combination thereof. It should be further understood that additional features may be added in the dual gate backside sensing FET sensor 200 for additional embodiments of the dual gate backside sensing FET sensor 200, and that some of the described features may be replaced or eliminated.

Fig. 3 depicts a schematic diagram of a portion of an example addressable array 300 of bio fet sensors 304 connected to bit lines 306 and word lines 308. It should be noted that the terms bit line and word line are used herein to indicate similarity to the configuration of an array in a memory device, however, it is not implied that the memory device or memory array must be included in the array. The addressable array 300 may have similarities to that employed in other semiconductor devices, such as Dynamic Random Access Memory (DRAM) arrays. For example, the dual gate backside sense FET sensor 200 described above with reference to fig. 2 may be formed in a location where a capacitor will be found in a DRAM array. The schematic diagram 300 is merely exemplary and one will recognize that other configurations are possible.

According to some embodiments, the bioFET sensors 304 may each be substantially similar to the dual gate backside sense FET sensor 200. The FET 302 is configured to provide an electrical connection between the drain terminal of the bio FET sensor 304 and the bit line 306. In this way, FET 302 is similar to an access transistor in a DRAM array. In some embodiments, the bioFET sensor 304 is a dual gate backside sense FET sensor and each includes: a sense gate provided by a receptor material disposed on a dielectric layer that covers a FET channel region disposed at a reaction site; and a control gate provided by a gate electrode (e.g., polysilicon) disposed on a dielectric layer overlying the FET channel region.

The addressable array 300 shows an array formation designed to detect small signal changes provided by biomolecules or biological entities introduced to the bio fet sensor 304. The array format using bit lines 306 and word lines 308 allows for a smaller number of input/output pads because the common terminals of different FETs in the same row or column are tied together. The amplifier may be used to enhance signal strength to improve the detection capability of the device having the circuit arrangement of diagram 300. In some embodiments, when a voltage is applied to a particular word line 308 and bit line 306, the corresponding access transistor 302 will turn on (e.g., like a switch). When the gate of the associated bio FET sensor 304 (e.g., such as the back side gate 222 of the dual gate back side sensing FET sensor 200) has a charge affected by the presence of biomolecules, the threshold voltage of the bio FET sensor 304 changes, thereby modulating the current (e.g., I _ds ). Current (e.g. I _ds ) Or threshold voltage (V) _t ) The change in (c) may be used to indicate the detection of the relevant biomolecule or biological entity.

Referring to fig. 4, an exemplary schematic 400 is presented. The exemplary schematic 400 includes access transistors 302 and a bioFET sensor 304 arranged as an array 401 of individually addressable pixels 402. The array 401 may include any number of pixels 402. For example, array 401 may include 128×128 pixels. Other arrangements may include 256×256 pixels or a non-square array, such as 128×256 pixels.

Each pixel 402 includes an access transistor 302 and a bio fet sensor 304 along with other components that may include one or more heaters 408 and a temperature sensor 410. In this example, access transistor 302 is an n-channel FET. The n-channel FET 412 may also act as an access transistor for the temperature sensor 410. In some embodiments, the gates of FETs 302 and 412 are connected, but this is not required. Each pixel 402 (and its associated components) may be individually addressed using a column decoder 404 and a row decoder 406. In some embodiments, each pixel 402 has a size of about 10 microns by about 10 microns. In some embodiments, each pixel 402 has a size of about 5 microns by about 5 microns or has a size of about 2 microns by about 2 microns.

Column decoder 406 and row decoder 404 may be used to control the on/off states of both n-channel FETs 302 and 412 (e.g., voltages applied together to the gates of FETs 302 and 412 and voltages applied together to the drain regions of FETs 302 and 412). Turning on the n-channel FET 302 provides a voltage to the S/D region of the bioFET sensor 304. When the bioFET sensor 304 is on, current I _ds Flows through the bioFET sensor 304 and can be measured.

The heater 408 may be used to locally increase the temperature around the bio fet sensor 304. The heater 408 may be constructed using any known technique, such as forming a metal pattern with a high current flowing therethrough. The heater 408 may also be a thermoelectric heater/cooler, like a Peltier device. The heater 408 may be used, for example, during a particular biological test to denature DNA or RNA or to provide a binding environment for a particular biological molecule. The temperature sensor 410 may be used to measure the local temperature around the bio fet sensor 304. In some embodiments, a control loop may be generated to control the temperature using the heater 408 and feedback received from the temperature sensor 410. In some embodiments, the heater 408 may be a thermoelectric heater/cooler that allows for localized active cooling of components within the pixel 402.

Referring to fig. 5, a cross section of an example dual gate backside sense FET sensor 500 is provided in accordance with some embodiments. The dual gate backside sense FET sensor 500 is one embodiment of the dual gate backside sense FET sensor 200. Accordingly, the foregoing elements from fig. 2 are labeled with the element numbers from fig. 2 and the description thereof will not be repeated herein. The dual gate backside sense FET sensor 500 includes a gate 202, a source region 204, a drain region 206, and a channel region 208, wherein the source region 204 and the drain region 206 are formed within a substrate 214. The gate 202, source region 204, drain region 206, and channel region 208 form a FET. It should be noted that the various components of fig. 5 are not intended to be drawn to scale and are exaggerated for visual convenience, as will be understood by those of skill in the art.

In some embodiments, the dual gate backside sense FET sensor 500 is coupled to various layers of metal interconnects 502 that form electrical connections with various doped regions and other devices formed within the substrate 214. The metal interconnect 502 may be fabricated using fabrication processes well known to those skilled in the art.

The dual gate backside FET sensor 500 may include a body region 504 separate from the source region 204 and the drain region 206. The body region 504 may be used to bias the carrier concentration in the channel region 208 between the source region 204 and the drain region 206. In some embodiments, a negative voltage bias may be applied to the body region 504 to improve the sensitivity of the dual gate backside FET sensor 500. In some embodiments, body region 504 is electrically connected to source region 204. In some embodiments, body region 504 is electrically grounded.

The dual gate backside FET sensor 500 may be coupled to additional circuitry 506 fabricated within the substrate 214. The circuit 506 may include any number of MOSFET devices, resistors, capacitors, and/or inductors to form a circuit that facilitates operation of the dual gate backside sense FET sensor 500. The circuit 506 may represent a readout circuit used to measure a signal from the dual gate backside FET sensor 500 indicative of analyte detection. The circuit 506 may include an amplifier, an analog-to-digital converter (ADC), a digital-to-analog converter (DAC), a voltage generator, logic circuitry, and/or DRAM memory, to name a few examples. In some embodiments, the circuit 506 includes digital components and does not measure analog signals from the dual gate backside FET sensor 500. All or some of the components of the additional circuitry 506 may be integrated in the same substrate 214 as the dual gate backside FET sensor 500. It should be appreciated that numerous FET sensors, each substantially similar to the dual gate backside FET sensor 500, may be integrated in the substrate 214 and coupled to additional circuitry 506. In another example, all or some of the components of the additional circuitry 506 are provided on another semiconductor substrate separate from the substrate 214. In yet another example, some components of the additional circuitry 506 are integrated in the same substrate 214 as the dual gate backside FET sensor 500, and some components of the additional circuitry 506 are provided on another semiconductor substrate separate from the substrate 214.

Still referring to the illustrative example of FIG. 5, the dual gate backside sense FET sensor 500 includes a semiconductor layer deposited on an isolation layer

An interface layer 508 within the opening above and above the channel region 208. In some embodiments, interface layer 508 has about 20 +.>

And about

And a thickness therebetween. The interface layer 508 may be a high-k dielectric material such as hafnium silicate, hafnium oxide, zirconium oxide, aluminum oxide, tantalum pentoxide, hafnium dioxide-aluminum oxide (HfO) ₂ -Al ₂ O ₃ ) An alloy, or any combination thereof. The interface layer 508 may serve as a support for the attachment of capture reagents, as will be discussed in more detail in the paragraphs for biosensing later. Solution 512 is provided over the reaction sites of dual gate backside sense FET sensor 500, and fluid gate 510 is disposed within solution 512. Solution 512 may be a buffer solution containing a capture reagent, target analyte, wash solution, or any other biological or chemical species.

An example operation of the dual gate backside FET sensor 500 as a pH sensor will now be described with reference to fig. 5.

Briefly, the fluid gate 510 is used to provide a dual gate backThe electrical contact of the "back gate" of the side FET sensor 500. Solution 512 is provided over the reaction sites of dual gate backside FET sensor 500, and fluid gate 510 is disposed within solution 512. The pH of the solution is generally the same as the hydrogen ions [ H ] in the solution ⁺ ]Is related to the concentration of (c). Accumulation of ions near the surface of interface layer 508 over channel region 208 affects the formation of an inversion layer within channel region 208, which forms a conductive path between S/ D regions 204 and 206. In some embodiments, the current I _ds Flows from one S/D zone to another S/D zone.

Measurable current I _ds To determine the pH of solution 512. In some embodiments, the fluid gate 510 serves as the gate of the transistor during sensing, while the gate 202 is left floating. In some embodiments, the fluid gate 510 serves as the gate of the transistor during sensing, while the gate 202 is biased at a given potential. For example, depending on the application, the gate 202 may be biased at a potential between-2V and 2V, while the fluid gate 510 is swept between voltage ranges. In some embodiments, the fluid gate 510 is biased at a given potential (or ground), while the gate 202 serves as the gate of a transistor during sensing (e.g., its voltage is swept across a range of potentials). The fluid gate 510 may be formed of platinum or may be formed of any other commonly used material(s) for a reference electrode in an electrochemical analysis. An example of a reference electrode is a silver/silver chloride (Ag/AgCl) electrode having a stable potential value of about 0.230V.

Fig. 6A shows ions in solution bound to the surface of interface layer 508. The highest atomic layer of interface layer 508 is depicted as various dangling O ^- ]、[OH][ OH ₂ ⁺ ]A key. As ions accumulate on the surface, the total surface charge affects the threshold voltage of the transistor. As used herein, the threshold voltage is the minimum potential between the gate and source of the FET sensor that is required to form a conductive path for minority carriers between the source and drain of the FET sensor. The total charge is also directly related to the pH of the solution, since higher positive charge accumulation indicates a lower pH and higher negative charge accumulation indicates a higher pH.

Fig. 6B shows an example change in threshold voltage due to different pH values in an n-channel FET sensor. As can be observed in this example, a 59mV increase in threshold voltage roughly represents a pH increase of 1 for the solution. In other words, a 1 pH change results in an equivalent 59mV total surface charge when measured as the voltage required to turn on the transistor.

Changing the threshold voltage of the dual gate backside FET sensor 500 also changes the time required to form a conductive path between the S/ D regions 204 and 206 for a given voltage input to the fluid gate 510 or gate 202. According to some embodiments, this time delay of "turning on" the FET sensor may be quantified using digital circuitry and used to determine the analyte concentration.

FIG. 7 depicts an example biosensing test using a dual gate backside FET sensor 500 to determine the local concentration of captured cells, according to some embodiments. Capture reagent 704 may be bound to dielectric layer 508 using binding molecules 702. The linking molecule 702 may have a reactive chemical group that binds to a portion of the dielectric layer 508. Examples of linking molecules include thiols. The surface of the dielectric layer 508 may also be silanized, or by exposing the surface of the dielectric layer 508 to ammonia (NH) ₃ ) Plasma to form reactive NH on a surface ₂ The groups form a linking molecule. The silylation process involves sequentially exposing the surface of the dielectric layer 508 to different chemicals to accumulate covalently-bound molecules on the surface of the dielectric layer 508, as is commonly understood by those skilled in the art. The capture reagent 704 may comprise an antibody that binds to a protein on the outer surface of the target cell 706 to be captured.

According to some embodiments, target cells 706 produce chemicals that can alter the pH of the surrounding solution that can be detected by bio fet sensor 500, as discussed above with reference to fig. 6A and 6B. In other embodiments, a solution 701 is introduced that comprises a matrix material (e.g., glucose) that is broken down by enzymes within target cells 706 to produce specific byproducts. An example by-product may be an acidic metabolite produced by glycolysis of glucose and the citric acid cycle (TCA). One specific example of such an acidic metabolite is positively charged poly (gamma-glutamic acid), thus altering the pH of the surrounding solution and signaling the presence of target cells 706. The overall chemical pathway for the final production of poly (gamma-glutamic acid) by the breakdown of glucose is depicted in fig. 8. It should be understood that other chemical pathways may also be used to produce other pH altered byproducts.

bioFET array embodiments

Fig. 9 depicts a top view of an example sensor array 902 having a plurality of bioFET sensors arranged in a repeating pattern. The bioFET sensors arranged in the sensor array 902 may each be an example of the FET sensor 500 described above with reference to fig. 5. A cross-sectional view taken across bioFET sensors 904 and 906 is depicted at the top left of the figure. Although only a particular number of bio fet sensors are depicted in the sensor array 902, it should be understood that the sensor array 902 may include any number of bio fet sensors and that the arrangement of sensors is not limited to organized rows and columns.

Each of the bio fet sensors 904 and 906 includes corresponding wells 905 and 907 that may be patterned by forming openings through the thickness of the isolation layer 210. According to some embodiments, each well in the sensor array 902 is substantially aligned over a channel region of a corresponding dual gate backside sense FET sensor. In the depicted example, well 905 is aligned over channel region 208a of bio fet sensor 904 and well 907 is aligned over channel region 208b of bio fet sensor 906. According to some embodiments, each of the patterned wells across the sensor array 906 has any size between 500nm×500nm and 500 μm×500 μm. The size of the patterned wells between these dimensions can help minimize the tradeoff between effective sensing of each bio fet sensor and the number of different target analytes detected by the sensor array 902. According to some embodiments, the spacing between each of the wells across the sensor array 906 is between 1 μm and 1 mm. Although not explicitly depicted in fig. 9 for clarity, the sensor array 902 may also include microfluidic channels coupled to its surface such that fluid may be delivered to each of the bio fet sensors in the sensor array 902 via the microfluidic channels.

The bio fet sensor 904 includes a patterned dielectric layer 908a within the well 905 and over the channel region 208 a. The bio fet sensor 906 similarly includes a patterned dielectric layer 908b within the well 907 and over the channel region 208 b. Dielectric layers 908a and 908b may be portions of the same deposited dielectric layer, or may be layers having the same material composition but deposited at different times. In other embodiments, dielectric layers 908a and 908b comprise different materials.

Other components of the sensor array 902 include a plurality of interconnect layers (not shown) to form electrical connections with the source/drain regions 204/206 and gates of each of the bio fet sensors in the array. In the illustrated example, the gates 202a and 202b are formed over the surfaces of the channel regions 208a and 208b, respectively. According to some embodiments, the gates 202a and 202b are formed on the surfaces of the channel regions 208a and 208b that are opposite the surfaces of the channel regions 208a and 208b having the dielectric layers 908a and 908b. Any surface of channel regions 208a and 208b should also be understood as a surface of semiconductor substrate 214. According to some embodiments, a carrier substrate 612 is included to provide mechanical stability and rigidity to the sensor array 902.

In some embodiments, isolation regions 914 are formed between adjacent bioFET sensors to reduce electrical cross-talk between the sensors. Isolation regions 914 may represent standard Shallow Trench Isolation (STI) structures filled with oxide.

Each bio fet sensor of the sensor array 902 may be individually addressable such that sensing may occur independently at any of the bio fet sensors in the array. In this way, multiple different analytes can be detected using the same sensor array 902 with different capture reagents bound to the dielectric layers of different bio fet sensors.

Fig. 10 shows a sensor array 902 in which some bio fet sensors contain capture reagents and other bio fet sensors do not. For example, the bioFET sensor 1002 does not contain any capture reagent, while the bioFET sensor 1004 contains capture reagent 1006 bound to its corresponding dielectric layer (as seen in cross section taken across line A-A'). Any number of bio fets in the sensor array 902 may be functionalized with capture reagent 1006, and similarly, any number of bio fet sensors in the sensor array 902 do not have capture reagent. In some embodiments, bio fet sensors without capture reagent may be used to provide control signals for those bio fet sensors that do have capture reagent 1006. Capture reagent 1006 may be deposited over portions of sensor array 902 using a variety of possible techniques, an example of which will be described later with reference to fig. 10.

Once the capture reagent 1006 has been placed on the various bio fet sensors, a target solution containing the target analyte 1008 to be detected or counted can be introduced over the sensor array 902. For example, the target analyte 1008 may comprise a particular cell that binds to the capture reagent 1006 after the target solution has been applied, as is a cancer cell, as depicted in section B-B' taken across the bio fet sensor 1004. In other examples, target analyte 1008 comprises any other type of microorganism. The number or density of target analytes 1008 bound to a particular bio fet sensor may be determined based on the measured drain current variations of the bio fet sensor. The exact sensing method for any given bio fet sensor will be discussed in more detail below with reference to fig. 14-15.

Fig. 11 depicts the array extension depicted in fig. 10 by adding additional capture reagents that bind to other bio fet sensors in the sensor array 902. Specifically, the bio fet sensor 1102 includes a capture reagent 1104 (as depicted in fig. 11) that is different from the capture reagent 1006 on the bio fet sensor 1004. Capture reagent 1104 may be designed to bind to a different type of cell than capture reagent 1006. In other examples, capture reagent 1104 binds to any type of analyte other than the analyte bound to capture reagent 1006. As depicted in fig. 11, a first plurality of bio fet sensors may be functionalized with capture reagent 1006, while a second plurality of bio fet sensors may be functionalized with capture reagent 1104.

According to an embodiment, a target solution is introduced over a sensor array 902 containing various analytes (e.g., different cell types or different microorganisms) that may bind to the capture reagent 1006 of the bio fet sensor 1004, the capture reagent 1104 of the bio fet sensor 1102, or not bind to the set of capture reagents. In the illustrated example, the target analyte 1106 binds to the capture reagent 1104. Target analyte 1106 can comprise a particular cell (as a cancer cell) or any other type of microorganism. The number or density of target analytes 1106 bound to a particular bio fet sensor may be determined based on the measured change in drain current of the bio fet sensor. The exact sensing method for any given bio fet sensor is discussed in more detail below with reference to fig. 14-15. Due to the individually addressable nature of each bio fet sensor in the sensor array 902 and the ability to place different capture reagents on different bio fet sensors in the array, detection of multiple different analytes can occur simultaneously.

Detecting the presence of a given analyte can be used to provide a binary determination of whether the analyte is present in a target solution. In other embodiments, the sensor array 902 is used to provide a general count or concentration of a given analyte in a target solution. In yet other embodiments, the sensor array 902 is used to monitor the continuous growth of cells or microorganisms captured over a given bio fet sensor or sensors.

Fig. 12 depicts a sensor array 902 used to monitor the growth of cells or other microorganisms captured over a given bio fet sensor in the array, according to some embodiments. The bio fet sensor 1202 is one example of a plurality of bio fet sensors in the sensor array 902 that have captured the first cell population 1204 shown in section A-A' taken across the bio fet sensor 902. The first population of cells 1204 may be captured using a particular capture reagent present at one or more of the bioFET sensors in the sensor array 902.

A medium may be provided over the sensor array 902 to allow the first cell population 1204 to grow into the second cell population 1206 after a first duration. Section B-B' taken across bio fet sensor 902 depicts a higher cell population that comprises second cell population 1206. According to an embodiment, the increased production of positively charged byproducts (e.g., poly (gamma-glutamic acid)) from each cell of the second cell population 1206 may be detected by the bioFET sensor 1202, as discussed above with reference to fig. 6-8. The increase in measured drain current may be related to the growth rate or total population size of the target cells.

At a later time period, the second cell population 1206 grows into a higher third cell population 1208, as shown in section C-C' taken across the bio fet sensor 1202. The same medium may be used to continuously grow cells from the first cell population 1204 into a third cell population 1208. In another example, the culture medium flows continuously over the sensor array 902 such that it remains fresh. According to an embodiment, the measured drain current of the bio fet sensor 1202 increases with a corresponding increase in the population size of captured target cells. Depending on the type of captured cells, the corresponding changes in growth rate and measured drain current may be different. While this description focuses on the measurement of a single bio fet sensor 1202 in the sensor array 902, it should be understood that multiple bio fet sensors of the sensor array 902 may be measured together to provide a single signal indicative of the growth rate of captured target cells at each of the multiple bio fet sensors. Furthermore, by placing different capture reagents on different sets of bioFET sensors, the same sensor array 902 can be used to monitor the growth rate of a plurality of different cell types.

In some embodiments, the specific capture reagent is not used on the bioFET sensors of the sensor array 902, but rather the cells are placed over the surface of the sensor array 902 and allowed to grow unrestricted over the surface of the sensor array 902. In this way, as cells grow over time, the sensor array 902 can be used to monitor how cells spread across the surface of the sensor array 902 using a bioFET sensor.

Fig. 13 includes another top view of a sensor array 902 and depicts depositing different fluid droplets to immobilize different capture reagents on different bio fet sensors, according to some embodiments. For example, the first fluid droplet 1302 may be deposited such that it covers the first plurality of bioFET sensors 1304. The first fluid droplet 1302 can include a first plurality of capture reagents bound to a dielectric layer of a first plurality of bio fet sensors 1304. In one example, the first plurality of capture reagents comprises antibodies designed to bind to a particular type of cell or microorganism. The second fluid droplet 1306 may be deposited such that it covers the second plurality of bio fet sensors 1308. The second fluid droplet 1306 may contain a second plurality of capture reagents that is different from the first plurality of capture reagents. The second plurality of capture reagents are bound to the dielectric layer of the second plurality of bio fet sensors 1308. In one example, the second plurality of capture reagents comprises antibodies designed to bind to another specific type of cell or microorganism than captured by the first plurality of capture reagents. A third fluid droplet 1310 may be deposited such that it covers a third plurality of bio fet sensors 1312. The third fluid droplet 1310 may contain a third plurality of capture reagents that are different from the first or second plurality of capture reagents. The third plurality of capture agents is bound to the dielectric layer of the third plurality of bio fet sensors 1312. In one example, the third plurality of capture reagents comprises antibodies designed to bind to another specific type of cell or microorganism than captured by the first or second plurality of capture reagents. Any number of droplets may be used across the surface of the sensor array 902 to place different capture reagents across different sets of bioFET sensors.

According to some embodiments, each of droplets 1302, 1306, and 1310 are deposited simultaneously across sensor array 902. In other embodiments, each of droplets 1302, 1306, and 1310 are placed at different times. Each of droplets 1302, 1306, and 1310 may remain above their corresponding bio fet sensor for a given period of time to ensure that enough capture reagent binds to the bio fet sensor. The sensor array 902 may be washed with a buffer solution between depositing different droplets. According to some embodiments, the diameter of any of droplets 1302, 1306, and 1310 is between 50 μm and 150 μm.

In some embodiments, the bioFET sensors of sensor array 902 may be arranged in other array configurations (e.g., staggered array configurations) in lieu of the linear array configuration shown in fig. 9-13. In some embodiments, the bio fet sensors in a staggered array configuration may be used to accommodate a number of bio fet sensors that is greater than a linear array of bio fet sensors within the same area. In some embodiments, the bioFET sensors of sensor array 902 may be arranged in a cellular configuration instead of the linear array configuration shown in fig. 9-13.

FIG. 14 depicts an example method 1400 of detecting different target reagents using a sensor array according to some embodiments. Each of the bioFET sensors in the sensor array may be a dual gate backside FET sensor as depicted in fig. 5. It is to be understood that additional operations may be provided before, during, and after the method 1400, and that some of the steps described below may be replaced or eliminated for additional embodiments of the method.

The method 1400 begins at block 1402, where a first fluid droplet is deposited over a sensor array. The first fluid droplet comprises a first plurality of capture reagents, such as antibodies. The first plurality of capture reagents are bound to a backside surface of a first plurality of bio fet sensors in the sensor array exposed to the first fluid drop. The backside surface of each of the bio fet sensors may be within a corresponding patterned well that spans the surface of the sensor array. The first plurality of capture reagents in the first fluid droplet may be bound to a dielectric layer deposited on a backside surface of each of the first plurality of bio fet sensors. The first fluid droplet may remain above the first plurality of bio fet sensors for a given period of time to ensure adequate binding of the capture reagent.

Next, the method 1400 proceeds to block 1404, where a second fluid droplet is deposited over the sensor array. The second fluid droplet contains a second plurality of capture reagents, such as antibodies, that are different from the first plurality of capture reagents. The second plurality of capture reagents are bound to a backside surface of a second plurality of bio fet sensors in the sensor array exposed to the second fluid drop. The second plurality of bio fet sensors are each different from the first plurality of bio fet sensors such that different capture reagents do not bind to the same bio fet sensor. The second plurality of capture reagents in the second fluid droplets may be bound to a dielectric layer deposited on a backside surface of each of the second plurality of bio fet sensors. The second fluid droplet may remain above the second plurality of bio fet sensors for a given period of time to ensure adequate binding of the capture reagent.

According to an embodiment, the method 1400 proceeds to block 1406, where a target solution is provided over a sensor array. The target solution may be introduced by dropping a droplet containing the target solution also over the various bioFET sensors in the sensor array. In another embodiment, the target solution flows through the sensor array in a microfluidic channel coupled to a surface of the sensor array such that both the first plurality of bio fet sensors and the second plurality of bio fet sensors in the sensor array are contained in the same microfluidic channel.

The target solution contains a plurality of target analytes, such as target cells or microorganisms. The target analyte may bind to the first or second plurality of capture reagents to determine the presence of the target analyte in the target solution. For example, the measured drain current of a corresponding bio fet sensor with a captured target analyte increases with increasing concentration of the captured target analyte. Since multiple different capture reagents can be used across different bio fet sensors of a sensor array, multiple different target analytes can be sensed from a given target solution.

According to some embodiments, after binding the target analyte to its corresponding capture reagent in the sensor array, another solution containing a matrix material (e.g., glucose) is introduced over the sensor array. The captured cells and other types of microorganisms can break down glucose to produce positively charged byproducts that can be measured by the corresponding bioFET sensor as a function of drain current.

In block 1408, a first voltage is applied to the gates of the first plurality of bio fet sensors and then the induced drain current is measured from the first plurality of bio fet sensors. Similarly, in block 1410, a second voltage is applied to gates of a second plurality of bio fet sensors, and then the induced drain current is measured from the second plurality of bio fet sensors. The first applied voltage and the second applied voltage may have the same magnitude.

The method 1400 proceeds to block 1412, where it is determined whether the target analyte is present at the first or second plurality of bio fet sensors. As described above, if the measured drain current of the first or second plurality of bio fet sensors increases significantly beyond a baseline measurement (e.g., increases beyond variations caused by noise and standard measurement errors), then it may be determined that the target analyte is present at the bio fet sensor exhibiting the increased drain current. This determination can be made across all bio fet sensors in the sensor array simultaneously to test for the presence of any number of target analytes.

FIG. 15 illustrates an example method 1500 of detecting a growth rate of a target cell or microorganism using a sensor array according to some embodiments. Each of the bioFET sensors in the sensor array may be a dual gate backside FET sensor as depicted in fig. 5. It should be understood that additional operations may be provided before, during, and after the method 1500, and that some of the steps described below may be replaced or eliminated for additional embodiments of the method.

The method 1500 begins at block 1502, where a capture reagent is deposited over various bio fet sensors of a sensor array. The capture reagent may be deposited in one or more droplets that are disposed over or flow in a microfluidic channel across the various bio fet sensors. The capture reagent may comprise an antibody designed to bind to a particular type of cell or microorganism.

After the capture reagent has had sufficient time to effectively bind to the various bio fet sensors, the method proceeds to block 1504 where a target solution with target cells or microorganisms is introduced over the sensor array. The target solution may be introduced by dropping a droplet containing the target solution also over the various bioFET sensors in the sensor array. In another embodiment, the target solution flows through the sensor array in a microfluidic channel coupled to a surface of the sensor array. Cells or microorganisms present in the target solution bind to the capture reagent at the various bioFET sensors.

According to some embodiments, after binding the target cells or microorganisms to their corresponding capture reagents in the sensor array, another solution containing a matrix material (e.g., glucose) is introduced over the sensor array. The captured cells or microorganisms can break down glucose to produce positively charged byproducts that can be measured by the corresponding bio fet sensor as the drain current changes.

The method 1500 proceeds to block 1506, where a first voltage is applied to the gates of the various bio fet sensors with the captured cells or microorganisms. Application of the first voltage causes the bio fet sensor to turn on and conduct, providing a measurable drain current. At block 1508, a first drain current is measured from the various bio fet sensors, individually or collectively. According to some embodiments, the measured magnitude of the first drain current corresponds to a concentration of cells or microorganisms present at the bioFET sensor at a present time of application of the first voltage.

The method 1500 proceeds to block 1510, where a second voltage is applied to the gates of the various bio fet sensors with the captured cells or microorganisms. The second voltage may have the same magnitude as the first voltage and be applied to the gates of the various bio fet sensors at a later time than the first voltage. At block 1512, a second drain current is measured from the various bio fet sensors, individually or collectively, due to the application of a second voltage. Due to the time difference between the applied first voltage and the second voltage, the growth of the cell or microorganism will cause an increase in the measured second drain current compared to the measured first drain current. The difference between the first drain current and the second drain current may be compared at block 1514 to determine the growth characteristics of cells or microorganisms at the various bio fet sensors.

In some embodiments, instead of applying separate voltages to the gates of the various bio fet sensors at different times to monitor the growth of cells or microorganisms, a single voltage is continuously applied and drain current is continuously measured. In this way, the various bio fet sensors of the sensor array may provide real-time monitoring of the growth of cells or microorganisms.

General biological applications

The biofets of the present disclosure may be used to determine the presence or absence of a target analyte. In some aspects, a bioFET can detect and measure absolute or relative concentrations of one or more target analytes. biofets can also be used to determine the static and/or dynamic levels and/or concentrations of one or more target analytes, thereby providing valuable information related to biological and chemical procedures. biofets may further be used to monitor enzymatic reactions and/or non-enzymatic interactions, including but not limited to binding. As an example, biofets may be used to monitor enzymatic reactions in which substrates and/or reagents are consumed and/or reaction intermediates, byproducts, and/or products are produced. Examples of reactions monitored using the biofets of the present disclosure can be used, for example, to confirm nucleic acid synthesis of a nucleic acid sequence.

The type of target analyte used in embodiments of the present disclosure may have any nature provided that a capture reagent is present that selectively, and in some cases specifically, binds to the target analyte. The target analyte may be present in the test sample or may be generated, for example, after the test sample is contacted with the sensing layer of the dual gate backside sensing bio fet or in the case that other reagents in solution are contacted with the sensing layer of the dual gate backside sensing bio fet. Thus, the types of target analytes include, but are not limited to, hydrogen ions (protons) or other ionic species, nonionic molecules or compounds, metals, metal complexes, nucleic acids, proteins, lipids, polysaccharides, and small chemical compounds, such as sugars, drugs, pharmaceuticals, chemical combinatorial library compounds, and the like. The target analyte may be naturally occurring or may be synthesized in vivo or in vitro. The target analyte may indicate that a reaction or interaction has occurred, or that its progress has been indicated. However, the target analytes measured by a bioFET according to the present disclosure are not limited and may include any of a variety of biological or chemical substances that provide relevant information about biological or chemical procedures (e.g., binding events such as nucleic acid hybridization and other nucleic acid interactions, protein-nucleic acid binding, protein-protein binding, antigen-antibody binding, receptor-ligand binding, enzyme-matrix binding, enzyme-inhibitor binding, cellular stimulation and/or triggering, interactions of cells or tissues with compounds (e.g., drug candidates), and the like). It is to be understood that the present disclosure further contemplates detection of target analytes in the absence of a receptor, e.g., detection of PPi and Pi in the absence of a PPi or Pi receptor. Any binding or hybridization event that causes a change in the transconductance of the dual gate backside sensing bioFET changes the current flowing from the drain to the source of the sensor described herein and detectable according to some embodiments.

To detect various target analytes, the sensing surface of the dual gate backside sensing bio fet of the present disclosure may be coated with a capture reagent for the target analyte that selectively binds to the target analyte of interest or, in some cases, to the analyte genus to which the target analyte belongs. The capture reagent that selectively binds to the target analyte preferentially binds to the molecules of that analyte (i.e., its binding affinity for that analyte is greater than its binding affinity for any other analyte). The binding affinity for the analyte of interest may be at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold greater than its binding affinity for any other analyte of interest. In addition to the relative binding affinities, the capture reagent has a sufficiently high absolute binding affinity to effectively bind the target analyte of interest (i.e., it has sufficient sensitivity). Capture reagents used in the methods and systems of the present disclosure may have binding affinities in the femtomolar, picomolar, nanomolar, or micromolar range and may be reversible.

The capture reagent may have any property (e.g., chemical, nucleic acid, peptide, lipid, or a combination thereof). The present disclosure contemplates capture reagents that selectively bind to ionic species (whether anionic or cationic) as ionophores. In some embodiments, the ionophore is a capture reagent and the ions to which it binds are target analytes. Ionophores comprise, for example, art-recognized carrier ionophores derived from microorganisms (i.e., small liposoluble molecules that bind to specific ions). In some embodiments, the capture reagent is a silane, valinomycin or salinomycin and the ion to which it binds is potassium. In some embodiments, the capture reagent is Meng Ningsu, filomycin or SQI-Pr and the ion to which it binds is sodium. And in other embodiments, the capture reagent is ionomycin, calicheamicin (a 23187) or CA 1001 (ETH 1001) and the ion to which it binds is calcium. In other aspects, the present disclosure contemplates capture reagents that bind to more than one ion. For example, beauvericin can be used to detect calcium and/or barium ions, nigericin can be used to detect potassium, hydrogen and/or lead ions, and gramicidin can be used to detect hydrogen, sodium and/or potassium ions.

The test sample may be from a naturally occurring source or may be non-naturally occurring. Naturally occurring test samples include, but are not limited to, bodily fluids, cells, or tissues to be analyzed for diagnostic, prognostic, and/or therapeutic purposes. The test sample may comprise any of cells, nucleic acids, proteins, sugars, lipids, and the like. In various embodiments, the test sample may comprise a chemical or biological library to screen for the presence of agents having specific structural or functional properties. The sample may be liquid or dissolved in a liquid and have a small volume, and thus may be subjected to high-speed, high-density analysis, such as analyte detection using microfluidics.

Examples of biofets contemplated by the various embodiments discussed herein include, but are not limited to, chemfets (chemfets), ion Sensitive FETs (ISFETs), immuno FET (ImmunoFET), gene FETs (genfets or DNA-FETs), enzyme FETs (enfets), receptor FETs, cell-based FETs, cell-free FETs, and liquid biopsy FETs. Thus, the biofets described herein can be used to detect target analytes with capture reagents and thus define bioFET types that are not mutually exclusive. As a non-limiting example, a liquid biopsy FET may detect cell-free DNA and may also be referred to as a cell-free FET or DNA-FET. See, for example, sakata et al, "potential detection by mononucleotide polymorphism using genetic field effect transistors (Potentiometric Detection of Single Nucleotide Polymorphism by Using a Genetic Field-effect transistor)", "biochemistry (Chembiochem)," 6 (2005): 703-10; ulu et al, "field effect transistor device-based label-free all-electronic nucleic acid detection System (Labelfree fully electronic nucleic acid detection system based on a field-effect transistor device)", biosensor and bioelectronics (Biosens Bioelectron) "19 (2004): 1723-31; sakurai et al, "Real-time monitoring of DNA polymerase reaction by micro ISFET pH sensor (Real-time monitoring of DNApolymerase reactions by a micro ISFET pH sensor)", analytical chemistry (Anal Chem) 64.17 (1992): 1996-1997.

For example, some embodiments provide a method for detecting nucleic acids, the method comprising contacting a probe nucleic acid bound to a surface of a backside sensing layer of a dual gate backside sensing bio fet with a sample and detecting binding of nucleic acids from the sample to one or more regions of the probe nucleic acid. This nucleic acid detecting bioFET may also be referred to as a GenFET or a DNA-FET.

In other aspects, some embodiments provide a method for detecting a protein, the method comprising contacting a probe protein molecule bound to a surface of a backside sensing layer of a dual gate backside sensing bio fet with a sample and detecting binding of a protein from the sample to one or more regions of the probe protein molecule. GenFET and DNA-FET can be used to detect proteins.

In other aspects, some embodiments provide a method for detecting nucleic acids, the method comprising contacting a probe protein molecule bound to a surface of a backside sensing layer of a dual gate backside sensing bio fet with a sample and detecting binding of nucleic acids from the sample to one or more regions of the probe protein molecule. In still other aspects, some embodiments provide a method for detecting an antigen, the method comprising contacting a probe antibody bound to a backside sensing layer of a dual gate backside sensing bio fet with a sample and detecting binding of an antigen from the sample to one or more regions of the probe antibody. These protein or antibody binding biofets may also be referred to as immunofets.

In other aspects, some embodiments provide a method for detecting an enzyme matrix or inhibitor, the method comprising contacting a probe enzyme bound to a surface of a backside sensing layer of a dual gate backside sensing bio fet with a sample and detecting binding of a physical to one or more regions of the probe enzyme from the sample (or production of an enzyme product in the sample). In still other aspects, some embodiments provide a method for detecting an enzyme, the method comprising contacting an enzyme matrix or inhibitor bound to a surface of a backside sensing layer of a dual gate backside sensing bio fet with a sample and detecting binding of a physical to one or more enzyme matrices or inhibitors from the sample (or production of an enzyme product in the sample). This enzyme-based bioFET may also be referred to as EnFET.

In other aspects, some embodiments provide a method for detecting protein-small molecule (e.g., organic compound) interactions, the method comprising contacting a small molecule bound to a surface of a backside sensing layer of a dual gate backside sensing bio fet with a sample and detecting binding of a protein from the sample to one or more regions of a probe small molecule. In still other aspects, some embodiments provide a method for detecting nucleic acid-small molecule (e.g., organic compound) interactions, the method comprising contacting a small molecule bound to a surface of a backside sensing layer of a dual gate backside sensing bio fet with a sample and detecting binding of nucleic acid from the sample to one or more regions of a probe small molecule. In either detection method, the sample may comprise small molecules and the capture reagent bound to the surface of the backside sensing layer may be a nucleic acid or a protein. In other aspects, the target analytes of interest are heavy metals and other environmental contaminants, and/or the bioFET array is specifically configured to detect the presence of different contaminants. These small molecule or chemical sensing biofets may also be referred to as chemfets.

In other aspects, some embodiments provide a method for detecting hydrogen ions and/or h+ concentration changes (i.e., pH changes). These ion-sensing biofets may also be referred to as ISFETs.

The systems and methods described herein may also be used to assist in identifying and treating diseases. For example, some embodiments provide a method for identifying sequences associated with a particular disease or for identifying sequences associated with a response to a particular active ingredient or therapeutic or prophylactic agent, the method comprising contacting a capture reagent (e.g., a nucleic acid probe) bound to the surface of a backside sensing layer of a dual gate backside sensing bio fet with a sample and detecting binding of nucleic acid (e.g., comprising a variant or deleting nucleic acid otherwise contained in a corresponding wild-type nucleic acid sequence) from the sample to one or more regions of the capture reagent. These biofets may also be referred to as genfets, DNA-FETs or liquid biopsy FETs.

Further application

Several additional applications of the dual gate backside sensing biofets described herein are contemplated. For example, the sensing layer of the dual gate backside sensing bioFET provides real-time, label-free quantification and analysis for a variety of biological, chemical, and other applications, including but not limited to gene expression analysis, comparative Genomic Hybridization (CGH), array-based exon-enrichment procedures, protein sequencing, tissue microarrays, and cell culture. In some embodiments, the dual gate backside sensing bio fet may be used to screen samples for the presence or absence of a substance, including but not limited to body fluids and/or tissues such as blood, urine, saliva, CSF or lavage fluid or environmental samples such as water supply samples or air samples. For example, the array can be used to determine the presence or absence of a pathogen (e.g., a food-borne or infectious pathogen), such as a virus, bacterium, or parasite, based on the target gene, proteosome, and/or other element. The array may also be used to identify the presence or absence, or to characterize cancer cells or cells indicative of another condition or disorder in the subject. Additional applications for using the dual gate backside sensing biofets described herein include U.S. patent No. 8,349,167 (gene expression analysis, comparative Genomic Hybridization (CGH), array-based exon enrichment procedure (Gene expression analysis, comparative Genome Hybridization (CGH), array-based exon enrichment processes)); 8,682,592 (Non-invasive prenatal diagnosis (NIP D), DNA/RNA contamination, SNP identification (Non-Invasive Prenatal Diagnosis (NIP D), DNA/RNA contamination, SNP identification)); 9,096,899 (method of providing amplified and sequenced DNA in flow cells (Method of amplifying and sequencing DNA within a flow cell is provided)); 9,340,830 (analysis of tumor samples (Analyzing a tumor sample)); 9,329,173 (automated system for testing intestinal salmonella (Automated system for testing for Salmonella enterica bacteria)); 9,341,529 (method (Method for manufacturing a pressure sensor) for manufacturing a pressure sensor); U.S. published application 2015/0353920; no. 2015/0355129 (detection of chemical and biological substances in body fluids (Chemical and biological substances detection in bodily fluid)); 2016/0054312 (chemically differentiated sensor array (Chemically differentiated sensor array for sample analysis) for sample analysis); identification and molecular characterization of CTCs associated with neuroendocrine prostate cancer (NEPC) (Identification and molecular characterization of the CTCs associated with neuroendocrine prostate cancer (NEPC))) No. 2016/0040245.

In some embodiments, the dual gate backside sensing bioFET can obtain single cell gene expression profiles with one or more cells from a cell sample of interest, such as in a heterogeneous cell sample. These samples typically exhibit high variation in their gene/biomarker expression levels (e.g., due to cell circulation, environment, and random transcription/transformation mechanisms), even in individual cells with the same phenotype. The dual gate backside sensing bioFET is capable of interrogating the expression profile of each cell in the sample. In a particular aspect, the inventive method for single cell molecular profiling avoids the need to separate cells of interest from heterogeneous cell samples, where individual profiling is available at each dual gate backside sensing bioFET. Direct molecular profiling in heterogeneous cell samples is advantageous for clinical diagnostic and biomarker discovery applications. In particular aspects, the dual gate backside sensing bio fet is used for molecular profiling and cell subtype of heterogeneous raw or enriched diseased tissue and biological fluid samples, such as biopsy tumor samples, endothelial cells from cardiovascular disease samples, bone marrow samples, lymph node samples, lymph, amniotic fluid, brain samples with different neurological disorders, pulmonary pathology samples, and/or any other heterogeneous diseased tissue samples of interest. Thus, for example, dual gate backside sensing biofets are used for molecular profiling of normal biological tissues and biological fluid samples to elucidate mechanisms such as differentiation, immune response, cell-cell communication, or brain development.

In some embodiments, dual gate backside sensing biofets are used to obtain single cell expression profiles in Circulating Tumor Cells (CTCs). CTCs may originate from metastasis and may be recirculated through the blood stream and lymph to colonize different organs and/or primary tumors, thereby causing secondary metastasis. CTCs play a key role in metastatic spread of cancer. Thus, detection of CTCs in blood (liquid biopsies) or Dissemination of Tumor Cells (DTCs) in bone marrow can be used to monitor tumor stage and will improve identification, diagnosis and treatment of cancer patients with high risk of metastatic recurrence. See, for example, U.S. patent No. 9,340,830 (column 205, lines 61-64); 9,447,411 (column 21, lines 42 to 54); 9,212,977 (column 19, lines 56 to 67); 9,347,946 (column 9, lines 16 to 30). In some embodiments, dual gate backside sensing biofets are used to obtain expression and mutation profiles in cell samples comprising CTCs as well as non-target contaminating cell types (e.g., white blood cells). See, for example, U.S. patent No. 9,340,830 (column 1, lines 41-67); 9,447,411 (column 2, lines 41 to 55); 9,212,977 (column 2, lines 48 to 67; column 3, lines 1 to 10); and 9,347,946 (column 9).

In other embodiments, the dual gate backside sensing bio fets described herein may provide point-of-care, portable, and/or real-time diagnostic tools. For example, it may provide an electronic reading of an enzyme-linked immunosorbent assay (ELISA) or other assay to detect various chemical or biological substances. The dual gate backside sensing bio fet may be configured to convert or convert a biochemical binding event or reaction into a readable electrical signal. The indirect detection of free-diffusing, electronically active species generated at sites of binding chemical or biological species may be performed using a dual gate backside sensing bioFET. An electronic readout ELISA protocol can be used that is capable of producing an enzyme of an electronically active species. In some embodiments, RNA switches are used to detect metabolites. See, e.g., mironov, alexander s et al, "small molecule sensing by nascent RNA: mechanisms to control transcription in bacteria (Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria) "," cells (Cell) "111.5 (2002): 747-756; winkler, wade, ali Nahvi and Ronald R.Breaker's thiamine derivatives bind messenger RNA directly to regulate bacterial gene expression (Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression), "Nature 419.6910 (2002): 952-956". In some embodiments, a dual gate backside sensing bioFET array is used to measure the kinetics of the reaction and/or compare the activity of the enzyme (including the substrate, cofactor, or another functional group) for readout.

Other applications of dual gate backside sensing bioFET arrays involve the use of molecular recognition sites, where molecules that specifically recognize specific target molecules are identified or designed and applied to the surface of the array. Previous chemFET operation has demonstrated the ability of a single individual ISFET to discriminate between ions (e.g., potassium).

In some embodimentsFor example, dual gate backside sensing biofets are used to monitor the presence and/or amount of specific molecules, including environmental tests such as specific toxins and vital elements. This test can use a molecular recognition site to measure both contaminant gas and particulate contamination, where molecules specifically recognizing a particular target molecule are identified or designed and applied to the surface of the array. See, for example, brzozka et al, "enhanced efficacy of potassium CHEMFET optimized through polysiloxane membranes (Enhanced performance of potassium CHEMFETs by optimization of a polysiloxane membrane)", sensor and actuator, B: chemistry (Sensors and Actuators B.chemical) 18, 38-41 (1994); sibald et al, "mini-flow cell of ChemFET integrated circuit with four functions for simultaneous measurement of potassium, hydrogen, calcium and sodium ions" (a minisure flow-through cell with a four-function ChemFET integrated circuit for simultaneous measurements of potassium, hydrogen, calcium and sodium ions) ", analytical chemistry report (Analytica Chimica acta.)" 159, 47-62 (1984); transduction of selective recognition of heavy metal ions by chemically modified field effect transistors (CHEMFETs) (Transduction of selective recognition of heavy metal ions by chemically modified field effect transistors (CHEMFETs)), (journal of american chemistry (Journal of the American Chemical Society)) 114, 10573-10582 (1992). In some embodiments, the dual gate backside sensing bioFET may be used with a personal, portable, and wearable detector system. Such a system may act as an early warning device that indicates to the user that the level of pollution in their current local environment is at a level that may cause some discomfort to the user or even cause respiratory problems. This is particularly relevant for people suffering from respiratory or bronchial or asthmatic conditions, where the user needs to take the necessary precautions. Dual gate backside sensing biofets have the capability to detect individual gases (e.g., such as NO _X 、SO ₂ And/or CO) and/or the ability to monitor temperature and humidity. See U.S. published application 2014/0361901; 2016/0110234 (0117 ]]Segments). For example, the contamination sensor may be referred to as a gas field effect transistor (gasFET). For example, the gasFET may contain a material having exposure to the ambientAn atmospheric gate-metallized FET. Protons may diffuse to the metal gas interface as the gas is absorbed on the surface. This results in a dipole layer that affects the threshold voltage of the device.

In some embodiments, the dual gate backside sensing bioFET can be used in vivo by being introduced into the body (e.g., in the brain or other region subject to ion flux) and then analyzing the changes. For example, the electrical activity of a cell can be detected by ion flow. Thus, the bioFET array can be integrated onto a novel ion discrimination tissue probe. Other applications include cochlear prostheses and retinal and cortical implants, for example. See, e.g., humayun et al, visual Research 43, 2573-2581 (2003); normann et al, visual research 39, 2577-2587 (1999).

End language

Embodiments of a sensor array having a plurality of dual gate backside sensing biofets and methods of using the sensor array are described herein. According to some embodiments, a sensor array includes a semiconductor substrate, a first plurality of FET sensors, and a second plurality of FET sensors. The first plurality of FET sensors each include: a first channel region located between a source region and a drain region in the semiconductor substrate and under a gate structure placed on a first side of the first channel region; and a dielectric layer disposed on a second side of the first channel region opposite the first side of the first channel region. A first plurality of capture reagents is coupled to the dielectric layer over the first channel region. The second plurality of FET sensors each include: a second channel region between source and drain regions in the semiconductor substrate and under a gate structure disposed on a first side of the second channel region; and the dielectric layer is disposed on a second side of the first channel region opposite the first side of the first channel region. A second plurality of capture reagents is coupled to the dielectric layer over the second channel region. The second plurality of capture reagents is different from the first plurality of capture reagents. The first plurality of FET sensors and the second plurality of FET sensors are arranged in a two-dimensional array.

According to some embodiments, a method of using a sensor device includes depositing a first solution droplet containing a first plurality of capture reagents over a first plurality of FET sensors formed in a semiconductor substrate. The first plurality of capture reagents are bonded to a dielectric layer on a first surface of the semiconductor substrate in a first plurality of openings disposed over the first plurality of FET sensors. The method also includes depositing a second solution droplet containing a second plurality of capture reagents over a second plurality of FET sensors formed in the semiconductor substrate. The second plurality of capture reagents are bonded to the dielectric layer on the first surface of the semiconductor substrate in a second plurality of openings disposed over the second plurality of FET sensors. The second plurality of capture reagents is different from the first plurality of capture reagents. The method also includes introducing a target solution over the first plurality of FET sensors and the second plurality of FET sensors. The method also includes applying a first voltage to a plurality of first gate structures of the first plurality of FET sensors. The first gate structure is located on a second surface of the semiconductor substrate opposite the first surface of the semiconductor substrate. The method also includes applying a second voltage to a plurality of second gate structures of the second plurality of FET sensors. The second gate structure is located on a second surface of the semiconductor substrate opposite the first surface of the semiconductor substrate. The method includes determining the presence of one or more target analytes in the target solution based on application of at least one of the first voltage or the second voltage.

According to some embodiments, a method of using a sensor device includes depositing a solution containing a plurality of capture reagents over a plurality of FET sensors formed in a semiconductor substrate. The plurality of capture reagents are bonded to a dielectric layer on the first surface of the semiconductor substrate in a plurality of openings disposed over the plurality of FET sensors. The method also includes introducing a second solution over the plurality of FET sensors such that one or more cells in the second solution bind to the capture reagent bound to the dielectric layer of the plurality of FET sensors. The method includes applying a first voltage to a plurality of gate structures of the plurality of FET sensors. The plurality of gate structures is located on a second surface of the semiconductor substrate opposite the first surface of the semiconductor substrate. A first current response of the plurality of FET sensors is measured based on the application of the first voltage. The method also includes applying a second voltage to a plurality of gate structures of the plurality of FET sensors a given period of time after application of the first voltage and measuring a second current response of the plurality of FET sensors based on application of the second voltage. The method includes determining a growth characteristic of the one or more cells based on a comparison between the first current response and the second current response.

It should be understood that the detailed description section, rather than the abstract section, is intended to be used to interpret the claims. Summary the paragraphs may describe one or more, but not all, exemplary embodiments of the present disclosure as contemplated by the inventor(s), and are therefore not intended to limit the disclosure and appended claims in any way.

It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined in accordance with the following claims and their equivalents.

Symbol description

100. Biosensor system

102. Sensor array

104. Fluid delivery system

106. Reading circuit

108. Controller for controlling a power supply

200. Dual gate backside sensing Field Effect Transistor (FET) sensor

202. Control grid

202a gate

202b gate

204. Source region

206. Drain region

208. Channel region

208a channel region

208b channel region

210. Isolation layer

211. Interconnect region

212. An opening

214. Substrate and method for manufacturing the same

215. Intervening dielectric

216. Electrical contact

218. Electrical contact

220. Front side gate contact

222. Backside gate contact

300. Addressable array/schematic

302. Access transistor

304. Biological field effect transistor (bioFET) sensor

306. Bit line

308. Word line

400. Schematic diagram of the present invention

401. Array

402. Individually addressable pixels

404. Column decoder

406. Line decoder

408. Heater

410. Temperature sensor

412 N-channel FET

500. Dual gate backside sense FET sensor

502. Metal interconnect

504. Body region

506. Circuit arrangement

508. Interface layer

510. Fluid gate

512. Solution

612. Carrier substrate

701. Solution

702. Binding molecules

704. Capture reagent

706. Target cells

902. Sensor array

904 bioFET sensor

905. Trap

906 bioFET sensor

907. Trap

908a dielectric layer

908b dielectric layer

914. Isolation region

1002 bioFET sensor

1004 bioFET sensor

1006. Capture reagent

1008. Target analytes

1102 bioFET sensor

1104. Capture reagent

1106. Target analytes

1202 bioFET sensor

1204. First population of cells

1206. Second cell population

1208. Third cell population

1302. First fluid drop

1304. First plurality of bioFET sensors

1306. Second fluid drop

1308. Second plurality of bioFET sensors

1310. Third fluid droplet

1312. Third plurality of bioFET sensors

1400. Method of

1402. Frame (B)

1404. Frame (B)

1406. Frame (B)

1408. Frame (B)

1410. Frame (B)

1412. Frame (B)

1500. Method of

1502. Frame (B)

1504. Frame (B)

1506. Frame (B)

1508. Frame (B)

1510. Frame (B)

1512. Frame (B)

1514. Frame (B)

I _ds Electric current

Claims (20)

1. A sensor array, comprising:

a semiconductor substrate;

a first plurality of FET sensors, each of the first plurality of FET sensors comprising:

a first channel region between a first source region and a first drain region in the semiconductor substrate,

a first gate structure located on a first side of the first channel region,

a dielectric layer disposed on a second side of the first channel region opposite the first side of the first channel region exposing at least a first portion of the dielectric layer above the first channel region to a target solution, an

A first plurality of capture reagents coupled to the dielectric layer over the first channel region;

a second plurality of FET sensors, each of the second plurality of FET sensors comprising:

a second channel region between a second source region and a second drain region in the semiconductor substrate,

a second gate structure on a first side of the second channel region,

the dielectric layer being disposed on a second side of the second channel region opposite the first side of the second channel region such that at least a second portion of the dielectric layer located over the second channel region is exposed to the target solution,

A second plurality of capture reagents coupled to the dielectric layer over the second channel region, wherein the second plurality of capture reagents is different from the first plurality of capture reagents,

a controller coupled to a plurality of the first gate structures of the first plurality of FET sensors and a plurality of the second gate structures of the second plurality of FET sensors, the controller configured to apply a first voltage to the plurality of the first gate structures and configured to apply a second voltage to the plurality of the second gate structures; and

A readout circuit coupled to the first plurality of FET sensors and the second plurality of FET sensors, and configured to determine the presence of one or more target analytes in the target solution based on at least one of the first voltage and the second voltage,

wherein the first plurality of FET sensors and the second plurality of FET sensors are arranged in a two-dimensional array and are coupled to a common reference electrode.

2. The sensor array of claim 1, further comprising:

an insulating layer is disposed over a surface of the semiconductor substrate.

3. The sensor array of claim 2, wherein the insulating layer includes a plurality of openings through a thickness of the insulating layer, the plurality of openings arranged over the first channel region and the second channel region.

4. The sensor array of claim 3, wherein the dielectric layer is placed in the plurality of openings.

5. The sensor array of claim 3, wherein each of the plurality of openings has an area between 500 nm x 500 nm and 500 μιη x 500 μιη.

6. The sensor array of claim 1, wherein at least one of the first plurality of capture reagents and the second plurality of capture reagents comprises a compound selected from the list consisting of: RNA, DNA, antibodies, enzymes, proteins, and cells.

7. The sensor array of claim 1, wherein the dielectric layer comprises a high-k dielectric material.

8. The sensor array of claim 1, further comprising microfluidic channels arranged over the first plurality of FET sensors and the second plurality of FET sensors.

9. A sensing method, comprising:

depositing a first solution droplet containing a first plurality of capture reagents over a first plurality of FET sensors formed in a semiconductor substrate such that the first plurality of capture reagents bind to a dielectric layer on a first surface of the semiconductor substrate in a first plurality of openings arranged over the first plurality of FET sensors;

Depositing a second solution droplet containing a second plurality of capture reagents over a second plurality of FET sensors formed in the semiconductor substrate such that the second plurality of capture reagents bind to the dielectric layer on the first surface of the semiconductor substrate in a second plurality of openings arranged over the second plurality of FET sensors, the second plurality of capture reagents being different from the first plurality of capture reagents;

forming a fluid delivery system configured to introduce a target solution over the first plurality of FET sensors and the second plurality of FET sensors;

coupling a plurality of first gate structures of the first plurality of FET sensors to a controller configured to apply a first voltage to the plurality of first gate structures, wherein the plurality of first gate structures are located on a second surface of the semiconductor substrate opposite the first surface of the semiconductor substrate;

coupling a plurality of second gate structures of the second plurality of FET sensors to the controller, the controller configured to apply a second voltage to the plurality of second gate structures, wherein the plurality of second gate structures are located on a second surface of the semiconductor substrate opposite the first surface of the semiconductor substrate; and

The first plurality of FET sensors and the second plurality of FET sensors are coupled to a readout circuit configured to determine the presence of one or more target analytes in the target solution based on the application of at least one of the first voltage and the second voltage.

10. The method of claim 9, wherein the introducing the target solution comprises flowing the target solution through a microfluidic channel of the fluid delivery system.

11. The method of claim 9, wherein the first solution droplet and the second solution droplet each have a diameter of about 100 μιη.

12. The method of claim 9, further comprising binding the one or more target analytes to the first plurality of capture reagents or the second plurality of capture reagents.

13. The method of claim 12, further comprising introducing a solution having a compound that reacts with the one or more target analytes to produce byproducts.

14. The method of claim 13, wherein the presence of the byproduct adjacent to the first plurality of capture reagents or the second plurality of capture reagents alters a threshold voltage of the first plurality of FET sensors or the second plurality of FET sensors, respectively.

15. A method of manufacturing a biosensor system, the method comprising:

depositing a solution containing a plurality of capture reagents over a plurality of FET sensors formed in a semiconductor substrate such that the plurality of capture reagents bind to a dielectric layer on a first surface of the semiconductor substrate in a plurality of openings disposed over the plurality of FET sensors;

forming a fluid delivery system configured to introduce a second solution over the plurality of FET sensors such that one or more cells in the second solution bind to the capture reagent bound to the dielectric layer of the plurality of FET sensors;

coupling a plurality of gate structures of the plurality of FET sensors to a controller configured to apply a first voltage and a second voltage to the plurality of gate structures, wherein the plurality of gate structures are located on a second surface of the semiconductor substrate opposite the first surface of the semiconductor substrate, and wherein the second voltage is applied a given period of time after the application of the first voltage; and

The plurality of FET sensors are coupled to a readout circuit configured to measure first and second current responses of the plurality of FET sensors based on the application of the first and second voltages, wherein the readout circuit is further configured to determine a growth characteristic of the one or more cells based on a comparison between the first and second current responses.

16. The method of claim 15, wherein the introducing the second solution comprises flowing the second solution through a microfluidic channel of the fluid delivery system.

17. The method of claim 15, wherein the depositing comprises depositing the solution containing the plurality of capture reagents as droplets over the plurality of FET sensors.

18. The method of claim 15, further comprising binding the one or more cells to the plurality of capture reagents.

19. The method of claim 18, further comprising introducing a solution having a compound that reacts with the one or more target cells to produce a byproduct.

20. The method of claim 19, wherein the presence of the byproducts adjacent to the plurality of capture reagents alters a threshold voltage of the plurality of FET sensors.

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